The uptake of calcium into mitochondria is an important process for regulating mitochondrial function and fine- tuning cellular calcium signaling. However, cell stress and injury perturbs calcium homeostasis resulting in mitochondrial calcium overload and reactive oxygen species (ROS) production that initiate necrotic cell death mediated by cyclophilin D (CyD)-dependent mitochondrial permeability transition pore (MPTP) formation. This results in mitochondria depolarization, ATP depletion and plasma membrane rupture. Necrotic cell death occurs during ischemia/reperfusion injury as a result of oxygen/glucose deprivation followed by increased calcium and production of ROS during reoxygenation. CyD-dependent cell death is a regulated process that involves mitochondrial calcium overload, ROS production and activation of calcium/calmodulin-dependent protein kinase II (CaMKII). We identified a cell death pathway induced in cultured mouse lung fibroblasts by calcium overload that involves CyD-dependent MPTP formation and CaMKII activation. This pathway was unexpectedly inhibited by the serine hydrolase inhibitors pyrrophenone and the triazole urea compound KT195 but did not involve the known targets of these inhibitors, the lipases cytosolic phospholipase A2a (cPLA2a) and alphabetahydrolase 6 (ABHD6), respectively. Preliminary data demonstrated that the inhibitors also blocked necrotic cell death in human lung epithelial cells and human neuroblastoma cells in response to the environmental toxicants paraquat and rotenone, respectively. The serine hydrolase inhibitors blocked cell death by inhibiting mitochondrial calcium uptake and preventing MPTP formation. Mitochondrial calcium uptake that triggers cell death occurs by transfer of calcium from the endoplasmic reticulum (ER) to mitochondria at regions of the ER membrane that form close contacts with mitochondria (mitochondria-associated membranes, MAM). These regions are enriched in calcium signaling regulators such as CaMKII and the IP3 receptor, and enzymes involved in lipid synthesis and metabolism. The regulation of mitochondrial calcium uptake leading to cell death is not fully understood and there is a lack of pharmacological inhibitors as tools to study the process. We hypothesize that a serine hydrolase localized at MAM regulates mitochondrial calcium uptake, CaMKII activation and cell death. The primary goal of this R21 exploratory proposal is to identify the target of the serine hydrolase inhibitors by using innovative chemical proteomic approaches, and to elucidate its function. Our results have uncovered a novel pathway implicating a serine hydrolase in regulating necrotic cell death that has broad implications for understanding how perturbation of mitochondrial calcium contributes to disease pathogenesis from a wide range of injuries such as exposure to environmental toxicants, in response to drug toxicity and from ischemia/reperfusion injury that occurs during stroke and cardiac arrest.